Melanin Biosynthesis Pathway Research Articles

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis

Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

Phlorotannins Derived From the Brown Alga Colpomenia bullosa as Tyrosinase Inhibitors

Tyrosinase catalyzes hydroxylation of L-tyrosine and dehydrogenation of L-DOPA in the melanin biosynthesis pathway. Tyrosinase inhibitors have potential use as cosmetic whitening agents and for preventing seafood deterioration. In this report, tyrosinase inhibitors extracted from brown alga Colpomenia bullosa (Scytosiphonaceae, Scytosiphonales) were investigated. Inhibitory principles were isolated from the extract and identified as phlorotannins, phloroglucinol (1), diphlorethol (2), triphlorethol C (3), which have not been isolated in a free form previously, and fucophlorethol C (4). Compounds 3 and 4 have not been reported previously as tyrosinase inhibitors. Triphlorethol C (3) was the most potent tyrosinase inhibitor among the phlorotannins isolated, whereas isomeric fucophlorethol C (4) displayed the weakest inhibitory activity. The results suggest that molecular structures of phlorotannins strongly affect their tyrosinase inhibitory activity.

Genomic Analysis and Assessment of Melanin Synthesis in Amorphotheca resinae KUC3009.

This study reports the draft genome of Amorphotheca resinae KUC30009, a fungal isolate with promising industrial-scale melanin production potential. The mechanisms for melanin or melanin-related pigment formation of this strain were examined through bioinformatic and biochemical strategies. The 30.11 Mb genome of A. resinae contains 9638 predicted genes. Genomic-based discovery analyses identified 14 biosynthetic gene clusters (BGCs) associated with secondary metabolite production. Moreover, genes encoding a specific type 1 polyketide synthase and 4-hydroxynaphthalene reductase were identified and predicted to produce intermediate metabolites of dihydroxy naphthalene (DHN)-melanin biosynthesis pathway, but not to DHN-melanin. These findings were further supported by the detection of increased flaviolin concentrations in mycelia and almost unchanged morphologies of the culture grown with tricyclazole. Apart from this, the formation of melanin in the culture filtrate appeared to depend on the laccase-like activity of multi-copper oxidases. Simultaneously, concentrations of nitrogen-containing sources decreased when the melanin formed in the media. Interestingly, melanin formation in the culture fluid was proportional to laccase-like activity. Based on these findings, we proposed novel strategies for the enhancement of melanin production in culture filtrates. Therefore, our study established a theoretical and methodological basis for synthesizing pigments from fungal isolates using genomic- and biochemical-based approaches.

Melanin synthesis and regulation in vivo and commonly used melanin inhibitors from natural products and traditional Chinese medicine

Melanin is an important factor affecting human skin color. This study defines its synthetic pathways and regulatory pathways have the practical significance for the treatment of diseases caused by pigmentation problems, such as chloasma. Besides, it also provides a theoretical basis for screening out melanin inhibitors and developing whitening and freckle products. At present, the melanin inhibitors used in whitening and freckle products mainly come from natural products and traditional Chinese medicine. This article first introduces the melanin biosynthesis pathway with tyrosinase as the core, defines the synthesis, transport and catalytic activity of tyrosinase as the three key factors affecting melanin synthesis, and then reviews two common types of melanin inhibitors, namely tyrosinase synthesis inhibitors and tyrosinase inhibitors derived from natural products and traditional Chinese medicine. This article provides guidance for the development of new melanin inhibitors, and puts forward the idea for combining and synergizing inhibitors according to different mechanisms, in order to develop new whitening formulas.

Identification of toxic mold species through Raman spectroscopy of fungal conidia.

We use a 785 nm shifted excitation Raman difference (SERDS) technique to measure the Raman spectra of the conidia of 10 mold species of especial toxicological, medical, and industrial importance, including Stachybotrys chartarum, Penicillium chrysogenum, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, and others. We find that both the pure Raman and fluorescence signals support the hypothesis that for an excitation wavelength of 785 nm the Raman signal originates from the melanin pigments bound within the cell wall of the conidium. In addition, the major features of the pure Raman spectra group into profiles that we hypothesize may be due to differences in the complex melanin biosynthesis pathways. We then combine the Raman spectral data with neural network models to predict species classification with an accuracy above 99%. Finally, the Raman spectral data of all species investigated is made freely available for download and use.

Management of periorbital hyperpigmentation: An overview of nature-based agents and alternative approaches.

Periorbital hyperpigmentation (POH) is a common dermatological condition that presents as dark periorbital area beneath the lower eyelids, and it is commonly found in females belonging to the age group of 16 to 45 years. The data presented in this review include studies conducted on patients with a clinical/histological diagnosis of POH or melasma. Many diverse topical depigmenting agents comprising an array of naturally obtained actives such as arabinoxylans, α-arbutin, asiaticoside, azelaic acid, beta-carotene, boswellic acid, caffeine, chrysin, curcumin, cyanidin-3-glucoside, d-glucoronic acid, dihydrochalcone, dipalmitoyl-hydroxyprolene, fucoxanthin, genistein, glabridin, b-glucogallin, hyaluronic acid, lactic acid, lycopene, niacinamide, pycnogenol, retinol, salidroside, and xymenynic acid demonstrated significant benefits in the management of POH. An exhaustive literature search revealed that other techniques such as blepharoplasty, carboxytherapy, calcium hydroxylapatite fillers, tear trough implant, Q-switched ruby laser, medicated tattoo, fat transfer, micro-needling, chemical peels, nitrogen plasma skin regeneration, intense pulsed light, and radiofrequency have been evaluated and reported to be beneficial in the treatment of POH. The use of topical depigmenting agents is the most widely reported method in the clinical management of POH. Of these, α-arbutin, caffeine, cyanidin-3-glucoside, and dihydrochalcone are reported to exhibit significant benefits. Combination products containing a blend of actives are reported to be better than single active containing products. This review aims to provide a comprehensive perspective on the role of several topical actives in the modulation of melanin and tyrosinase biosynthesis pathway involved in the complex pathophysiology of POH. It also presents the advantages of combination products and other alternative therapies used in the management of POH.

Plant-based skin lightening agents: A review

Hyperpigmentation is a skin disorder that occurs widely in the human population in which darkened patches or spots appear on the skin. Cosmetic products containing natural skin lightening agents are regarded as a safe alternative and effective approach to this hyperpigmentation problem. The review paper aims to discuss on some potential plant species with skin lightening properties by focusing on the presence of responsible bioactive compounds and their specific mechanisms of action in minimizing melanin production in hyperpigmentation problem. The finding shows that different bioactive compounds have various modes of action on melanin biosynthesis pathway including tyrosinase inhibition, melanosomes transfer inhibition, anti-oxidant and anti-inflammatory activities. Some plants with skin lightening activity contain several bioactive compounds which contribute to an enhancement of skin lightening effects and inhibition of hyperpigmentation. In conclusion, the screening of potential skin lightening ingredients from natural sources is very crucial process before formulation development for an effective skin lightening product.

Palladium(II)-Catalyzed Efficient Synthesis of Wedelolactone and Evaluation as Potential Tyrosinase Inhibitor.

Tyrosinase is an enzyme widely distributed in nature, which has multiple functions, especially in the melanin biosynthesis pathway. Despite the few clinically available tyrosinase inhibitors for whitening, a great demand remains for novel compounds with low side effects in terms of potential carcinogenicity and improved clinical efficacy. A natural product, wedelolactone (WEL), with a polyhydroxyl moiety, attracted our attention as a potential tyrosinase inhibitor. Before we studied the biological activity of the natural product, a synthetic methodological research was firstly carried to obtain enough raw material. WEL could be obtained efficiently through palladium-catalyzed boronation/coupling reactions and 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ)-involved oxidative deprotection/annulation reactions. Immediately after, the natural product was proven to be an efficient tyrosinase inhibitor. In conclusion, we developed a mild and efficient approach for the preparation of WEL, and the natural product was disclosed to have anti-tyrosinase activity, which could be widely used in multiple fields.

Development of a novel fungicide, tolprocarb.

Tolprocarb developed by Mitsui Chemicals Agro, Inc. (Tokyo, Japan) was discovered as a new oomycete fungicide. However, its antifungal spectrum and action mechanisms against fungi are completely different from those of the original compound, iprovalicarb. Tolprocarb has a potent and highly controlled effect on a rice blast fungus Magnaporthe grisea, and its mode of action was revealed to be the inhibition of polyketide synthase in the melanin biosynthesis pathway. In addition, tolprocarb induced systemic acquired resistance in Arabidopsis thaliana and rice (Oryza sativa L.). Owing to these double modes of action, tolprocarb can effectively control not only rice blast but also bacterial diseases, and has a low risk of developing fungicide-resistant isolates. Tolprocarb also provides long-term residual activity. A meta-analysis was performed in order to demonstrate tolprocarb's superior control against panicle blast in paddy fields. In addition, tolprocarb did not show cross-resistance against the fungi that are resistant to dehydratase inhibitor in melanin biosynthesis (Melanin Biosynthesis Inhibitor-Dehydratase; MBI-D) or respiratory complex III: cytochrome bc 1 at Quinone outside site inhibitor (Quinone outside Inhibitor; QoI). Owing to its stable effects, tolprocarb appears to be a suitable choice for practical use against fungi in the rice production field.

Global downregulation of pigmentation-associated genes in human premature hair graying.

Premature hair graying, or canities, is a complex multi-factorial process with negative effects on affected individuals. The aim of the present study was to investigate the possible underlying mechanisms of premature hair graying at the genetic level. A total of 5 unrelated Han Chinese individuals presenting with premature hair graying (25–40 years old, with >1% hair affected) were enrolled in the present study. RNA sequencing was performed to identify gene expression changes between the follicular cells of grey and black hair from the cohort. A total of 127 differentially expressed genes (DEGs) were identified. These DEGs were overrepresented in categories associated with the pigmentation pathway, with a decreased expression of key genes responsible for melanin synthesis. Of note, the decreased expression of certain transcription factors and the increased expression of certain precursor microRNAs observed may explain for the downregulation of certain other DEGs, which were identified as their targets via Starbase v2 and Integrated Motif Activity Response Analysis. The DEGs were also enriched in terms associated with the nervous system, indicating that neural disturbances may also have certain roles in premature hair graying. Of note, five of the downregulated DEGs were associated with aging according to the JenAge Aging Factor Database. To the best of our knowledge, the present study was the first genome-wide survey of the gene expression profile associated with premature hair graying. Dysfunction of the melanin biosynthesis pathway is probably the direct cause of hair graying and the present results provide valuable clues for further functional and mechanistic investigation.

Oculocutaneous Albinism with Squamous Cell Carcinoma, Bowen's Disease and Actinic Keratosis: A Case Report

Oculocutaneous Albinism with Squamous Cell Carcinoma, Bowen's Disease and Actinic Keratosis: A Case Report

Pleiotropic Effects of ebony and tan on Pigmentation and Cuticular Hydrocarbon Composition in Drosophila melanogaster.

Pleiotropic genes are genes that affect more than one trait. For example, many genes required for pigmentation in the fruit fly Drosophila melanogaster also affect traits such as circadian rhythms, vision, and mating behavior. Here, we present evidence that two pigmentation genes, ebony and tan, which encode enzymes catalyzing reciprocal reactions in the melanin biosynthesis pathway, also affect cuticular hydrocarbon (CHC) composition in D. melanogaster females. More specifically, we report that ebony loss-of-function mutants have a CHC profile that is biased toward long (>25C) chain CHCs, whereas tan loss-of-function mutants have a CHC profile that is biased toward short (<25C) chain CHCs. Moreover, pharmacological inhibition of dopamine synthesis, a key step in the melanin synthesis pathway, reversed the changes in CHC composition seen in ebony mutants, making the CHC profiles similar to those seen in tan mutants. These observations suggest that genetic variation affecting ebony and/or tan activity might cause correlated changes in pigmentation and CHC composition in natural populations. We tested this possibility using the Drosophila Genetic Reference Panel (DGRP) and found that CHC composition covaried with pigmentation as well as levels of ebony and tan expression in newly eclosed adults in a manner consistent with the ebony and tan mutant phenotypes. These data suggest that the pleiotropic effects of ebony and tan might contribute to covariation of pigmentation and CHC profiles in Drosophila.

Expression of tyrosinase genes associated with fruiting body formation and pigmentation in Grifola frondosa

Chemical and physical properties (including ultraviolet spectrum) of a brown pigment isolated from Grifola frondosa fruiting bodies were almost identical to those of fungal melanin. From the full genome sequence of G. frondosa , genes involved in the melanin biosynthesis pathway of this polypore mushroom were identified, including tyrosinases ( tyr1 and tyr2 ). Transcriptional analyses showed dramatic changes only in the expression of tyr2 in the primordial stage. Immuno-electron microscopy using anti-tyrosinase antibodies showed that TYR2 localizes in the cell walls of primordia. Therefore, TYR2 may be closely associated with melanin biosynthesis in this polypore mushroom G. frondosa , and melanin might be produced in the cell wall. • Brown pigment from Grifola frondosa fruiting bodies was identified as melanin. • We identified tyrosinases tyr1 and tyr2 from the full genome sequence of G. frondosa. • Transcription of tyr2 showed dramatic increase in the primordial stage only. • TYR2 localized in the cell wall of wild-type primordial cells.

Relationship of the orange tissue morphotype with shell and pearl colouration in the mollusc Pinctada margaritifera

Molluscs display a vast range of shell colours both between and within species. However, only a few species show colour variation in their soft tissues. In French Polynesia, the pearl oyster Pinctada margaritifera has three tissue morphotypes: the black wild-type and two rare mutations: white albino and orange mantle. Phenotypic transmission is known to occur from these phenotypes when they are used as graft donors for pearl production, leading to multicoloured and white pearls from black and albino mantle grafts, respectively. The present study furthers this knowledge by examining the phenotypic association between the orange mantle tissue morphotype and hard tissues: shells and cultured pearls. Based on a large experimental graft, shell colour quantification and pearl qualification showed that the orange morphotype is associated with light-coloured shells and pearls. Expression analysis of some candidate genes previously identified in the white mantle mutant, tested here on both graft and pearl sac tissues from orange mantle donors, confirmed the involvement of genes associated with shell matrix protein (shem4) and the melanin biosynthesis pathway (zinc). This study provides fundamental information on the mechanism behind mantle tissue colour in P. margaritifera and its association with biomineralisation and pigmentation processes that will be potentially valuable in future selection programs.

Transcription factor CgAzf1 regulates melanin production, conidial development and infection in Colletotrichum gloeosporioides.

Rubber anthracnose caused by Colletotrichum gloeosporioides leads to huge economic loss in the natural rubber industry every year. Conidia of C. gloeosporioides are a major infection source but little is known about molecular mechanisms underlying conidial development and infection. In this study, the C. gloeosporioide C2H2 zinc-finger protein transcription factor gene CgAzf1 is shown to be involved in melanin production, conidial development and infection. Deletion of CgAzf1 resulted in decreased melanin production and hydrophilicity of aerial mycelium was increased. The mutants also showed reduced conidiation, low germination rate, and the formation of appressorium lagged too. Virulence assays showed that the CgAzf1 deletion strain could not infect intact rubber tree leaves and had an attenuated virulence on the wounded leaves. Quantitative RT-PCR showed that CgAzf1 regulates expression of genes involved in the MAPK, cAMP-PKA and melanin biosynthesis pathways.

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi.

SummaryPerylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.

Development of an Expression Vector to Overexpress or Downregulate Genes in Curvularia protuberata.

Curvularia protuberata, an endophytic fungus in the Ascomycota, provides plants with thermotolerance only when it carries a mycovirus known as Curvularia thermotolerance virus (CThTV), and forms a three-way symbiotic relationship among these organisms. Under heat stress, several genes are expressed differently between virus-free C. protuberata (VF) and C. protuberata carrying CThTV (AN). We developed an expression vector, pM2Z-fun, carrying a zeocin resistance gene driven by the ToxA promoter, to study gene functions in C. protuberata to better understand this three-way symbiosis. Using this new 3.7-kb vector, five genes that are differentially expressed in C. protuberata—including genes involved in the trehalose, melanin, and catalase biosynthesis pathways—were successfully overexpressed or downregulated in VF or AN C. protuberata strains, respectively. The VF overexpression lines showed higher metabolite and enzyme activity than in the control VF strain. Furthermore, downregulation of expression of the same genes in the AN strain resulted in lower metabolite and enzyme activity than in the control AN strain. The newly generated expression vector, pM2Z-fun, has been successfully used to express target genes in C. protuberata and will be useful in further functional expression studies in other Ascomycota fungi.

Comparative proteomic analysis between nitrogen supplemented and starved conditions in Magnaporthe oryzae

BackgroundFungi are constantly exposed to nitrogen limiting environments, and thus the efficient regulation of nitrogen metabolism is essential for their survival, growth, development and pathogenicity. To understand how the rice blast pathogen Magnaporthe oryzae copes with limited nitrogen availability, a global proteome analysis under nitrogen supplemented and nitrogen starved conditions was completed.MethodsM. oryzae strain 70–15 was cultivated in liquid minimal media and transferred to media with nitrate or without a nitrogen source. Proteins were isolated and subjected to unfractionated gel-free based liquid chromatography-tandem mass spectrometry (LC-MS/MS). The subcellular localization and function of the identified proteins were predicted using bioinformatics tools.ResultsA total of 5498 M. oryzae proteins were identified. Comparative analysis of protein expression showed 363 proteins and 266 proteins significantly induced or uniquely expressed under nitrogen starved or nitrogen supplemented conditions, respectively. A functional analysis of differentially expressed proteins revealed that during nitrogen starvation nitrogen catabolite repression, melanin biosynthesis, protein degradation and protein translation pathways underwent extensive alterations. In addition, nitrogen starvation induced accumulation of various extracellular proteins including small extracellular proteins consistent with observations of a link between nitrogen starvation and the development of pathogenicity in M. oryzae.ConclusionThe results from this study provide a comprehensive understanding of fungal responses to nitrogen availability.

Genome sequence of the ectophytic fungus Ramichloridium luteum reveals unique evolutionary adaptations to plant surface niche

BackgroundEctophytic fungi occupy the waxy plant surface, an extreme environment characterized by prolonged desiccation, nutrient limitation, and exposure to solar radiation. The nature of mechanisms that facilitate adaptation to this environment remains unclear. In this study, we sequenced the complete genome of an ectophytic fungus, Ramichloridium luteum, which colonizes the surface of apple fruit, and carried out comparative genomic and transcriptome analysis.ResultsThe R. luteum genome was 28.18 Mb and encoded 9466 genes containing 1.85% repetitive elements. Compared with cell-penetrating pathogens, genes encoding plant cell wall degrading enzymes (PCWDEs), PTH11-like G protein-coupled receptors (GPCRs) and effectors were drastically reduced. In contrast, genes encoding cutinases and secretory lipases were strikingly expanded, and four of nine secretory lipases were probably acquired by horizontal gene transfer from Basidiomycota. Transcriptomic analysis revealed elevated expression of genes involved in cuticle degradation (cutinase, secretory lipase) and stress responses (melanin biosynthesis, aquaporins, lysozymes and HOG pathway).ConclusionsTaken together, our results highlight genomic features associated with evolution of surface niche adaptation by the ectophytic fungus R. luteum, namely the contraction of PCWDEs, PTH11-like GPCRs and effectors, and the expansion of cuticle degradation and stress tolerance.

The selective cytotoxicity of new triazene compounds to human melanoma cells

Metastatic melanoma still remains one the most difficult cancers to overcome. The aim of our research was the design of anti-tumour triazene compounds 3 for application to a melanoma-specific therapy. The strategy exploits the unique enzyme pathway of melanin biosynthesis for conversion of non-toxic prodrugs into toxic drugs in the melanoma cell. The compounds 3 were designed by coupling two active moieties, the alkylating triazenes and different tyrosinase substrates. All compounds 3 revealed to be chemically stable in isotonic phosphate buffer (PBS) at physiologic pH (t½≥48h), and most of them showed to be slowly hydrolysed in human plasma (1.5≤t½ (h)≤161). Compounds 3c–n revealed to be excellent tyrosinase substrates (0.74≤t½ (min)≤6) with the best tyrosinase substrate 3l releasing MMT 45s after tyrosinase activation. Structure-activity relationship studies allowed the identification of the better structural features for enzyme affinity. Furthermore, the derivatives 3l and 3m showed cell selectivity with significant cytotoxic effects (IC50 values of 46–65μM) against melanoma cell lines with tyrosinase overexpression MNT-1 and B16F10.